A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy

Abstract

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Figure 1. The Python mutation leads to dilated cardiomyopathy.

(A) A 13-week-old Python mouse compared to a littermate control. (B) Kaplan-Meier analysis of onset of overt CHF in Python mice on the C3H/HeN and C57BL/6J genetic backgrounds compared to wild type littermate controls. (C) Photo of the excised hearts from a 13-week-old Python and wild type mouse. Note the grossly enlarged ventricles and atria. (D) H&E section through a wild type and Python heart showing showing clinical signs of CHF. Note the distended ventricles; R = right, L = left. (E) Oedematous dermal connective tissue evident in a H&E-stained section from a Python mouse showing clinical signs of CHF compared to a littermate control. (F) MSB staining of heart sections of Python mice showing clinical signs of CHF. Note enlarged cardiomyocytes (indicated by red bracket) and increased collagen deposition (indicated by black bracket). (G) Morphometric analysis of MSB-stained section demonstrating the level of increase in collagen deposition. Data shown is % of image area (mean ± SEM) that is taken up by collagen deposition from 3 Py/+ showing overt signs of CHF and 3 +/+ age-matched males. Five images were taken per sample. (H) Evidence of cardiac calcification in a Von Kossa-stained section of a Python heart.

Figure 2. Genetic linkage analysis and positional cloning of the Py mutation.

(A) Genotypes of two informative recombinants exhibiting the Python phenotype. The mutation was localized to a 787 Kb interval (indicated by the bracketed region). (B) Sequence of part of the 11^th exon of the Dnm1l gene reveals a G–T substitution in the Python allele. The reading frame of part of exon 11 is shown with the amino acid substitution that results. (C) ClustalW2 alignment of part of domain M of vertebrate Dnm1l orthologues and the yeast Dnm1 homologue. Note that the cysteine residue is conserved in all species examined. Amino acid colours represent similarity groupings. (D) Alignment of the entire domain M of the mouse dynamins Dnm1l, Dnm1, Dnm2 and Dnm3. Identical or functionally very similar amino acids are shown grouped together by colour on the basis of size, charge and hydropathy. The arrow indicates the position of the cysteine altered by the Python mutation.

Figure 3. Effect of the Python mutation on the Dnm1l protein.

(A) Ribbon representation of the homology model of the dimeric form of Dnm1l protein in the extended conformation and embedded in a lipid membrane. The dimeric conformation model and coordinates of the lipid membrane were generated by the superposition of the structural model of human Dnm1l onto the structure of bacterial dynamin-like protein Bdlp2 . The positions of amino acid mutations reported in yeast Dnm1 and mammalian Dnm1l are shown as sphere representations in pink. A region that could not be modelled because of lack of a homologous region in BDLP is labelled with ‘a’. Colours indicate protein domains: GTPase (red), M domain (yellow) and GED (blue). (B) A helix wheel projection of the mutation-containing region of the Dnm1l protein. The hydropathy indices of the amino acids have been divided into relatively hydrophilic (blue) and relatively hydrophobic (red). An arrow indicates the cysteine that is substituted in the Py allele. Note how this face of the alpha-helix is relatively hydrophobic. (C) The locations within Dnm1l of the protein sequences used in the yeast two-hybrid analysis. (D) Yeast two hybrid analysis of all combinations of protein sequences as either bait (GAL4 DNA binding domain in pDEST32) and prey (GAL4 activation domain in pDEST22). Duplicate yeast colonies from each transfection grown on a –trp-leu plate (top), –trp-leu-his +100 mM AT (middle, colonies exhibiting growth are boxed in red) and a filter lift from the –trp-leu plate assayed for b-galactosidase activity (bottom). (E) Level of b-galactosidase in liquid cultures from interactors detected in (D) along with comparison to those with introduced Python mutation. Levels shown are relative to average of full-length wild type interaction. Each assay is the mean ± SEM of 6 independent measurements from 6 individual colonies for each combination.

Figure 4. Mitochondrial and peroxisomal morphology is altered by the Python mutation.

(A) Western blot analysis of total heart and liver protein extracts from 5-week-old male mice of the indicated genotypes demonstrating that Dnm1l protein levels are not altered. Western blots were re-examined using an anti-Tim23 antibody, an inner mitochondrial membrane protein, which demonstrate that the level of this mitochondrial protein is also not altered in Python mutants. After stripping, a-tubulin was detected to demonstrate loading levels of protein extracts. (B) Typical example of immunocytochemistry in neonatal mouse skin fibroblasts using an anti-DRP1 antibody. There was no appreciable difference between Python fibroblasts and wild type littermate controls. (C) Typical example of mitochondria (shown in red after staining with Mitotracker Orange) from cultured Python neonatal skin fibroblasts compared to littermate controls. (D) Typical example of peroxisomes (shown in green after incubation with an anti-catalase antibody and FITC-labelled secondary antibody) from cultured Python neonatal skin fibroblasts compared to littermate controls. The areas in C and D bounded by white squares are magnified and shown in the middle frames. Nuclei are stained with Hoechst 33258. (E) Typical example of FACS analysis of early passage skin fibroblasts from a Python neonate and littermate control after labelling with Mitotracker Orange. The histogram of fluorescence for both cells types is similar.

Figure 5. Homozygosity for the Py allele results in embryonic lethality.

(A) Distribution of genotypes of embryos recovered from Py/+ x Py/+ intercrosses. Embryo numbers genotyped at each time point were: E8.5–26, E9.5–48, E10.5–20, E11.5–19, E12.5 20, E13.5–21, E15.5–10 and E19.5–14. (B) Comparison of day 11.5 embryos heterozygous and homozygous for the Py allele. Homozygous embryos are growth retarded and have a severe posterior truncation. (C) Comparison of day 9.5 +/+, Py/+ and Py/Py embryos. Typical examples of individual fibroblasts obtained from culturing these embryos and stained with MitoTracker Orange are shown above. Below, typical examples of E9.5 embryonic fibroblasts in culture three days after embryo harvest. Note that the Py/Py cells have failed to proliferate and only a single cell is visible in this field.

Figure 6. Cardiomyocyte energy metabolism is altered in Python mice.

(A) Plasma lactate levels (mean ± SEM) in adult Python (n = 29) and wild type (n = 33) mice aged between 5 and 9 weeks and in mice. Sexes have been pooled. (B) The relative mitochondrial:nuclear DNA levels in heart samples from Python and control mice as assessed using Q-PCR. ‘Py/+ overt’ refers to heart samples taken from Python mice at the time of overt symptoms of heart failure. All other mice were aged between 5 and 7 weeks at time of sampling. *P<0.05, 1-way Anova with Bonferroni's Multiple Comparison Post-Test. (C) Typical example of enzyme histochemical staining for succinate dehydrogenase (SDH) and Complex IV from hearts of Python males suffering from overt CHF (aged 93 days) and an age-matched littermate control. Typical sections stained with Haematoxylin and Eosin (H&E) and Martius/Scarlet Blue (MSB) to stain connective tissue are shown for comparison. Note the scarring and loss of myocytes in Python hearts. (D) Comparison of Complex IV activity and quantity (mean ± SEM) measured in extracts from hearts of Python mice showing overt signs of CHF and wild type controls aged 91–103 days (n = 7 Python, n = 7 controls). (E) Representative transmission electron micrographs of Python and control hearts from mice aged 9–10 weeks. Scale bar = 2 µm. The graphs on the right summarizes the morphometric measurements (mean ± SEM) of mitochondrial area (reflecting volume per cell) and mitochondrial size in Python (4 mice; n = 75 micrographs) and wild type (4 mice; n = 55 micrographs) heart EM images. **P<0.01. (F) Mitochondrial respiratory complex enzyme activities (mean ± SEM measured in extracts of heart tissue from Python (n = 8) and control mice (n = 8) aged 10 weeks and normalized to level of citrate synthase activity. All activities expressed as nmol/min/mg total protein except complex IV which is expressed as k/min/mg. Complex I: NADH: Ubiquinone reductase, Complex II–III: Succinate: Cytochrome c reductase, Complex IV: Cytochrome c oxidase. (G) Overall citrate synthase activity (mean ± SEM nmol/min/mg total protein) in heart extracts from H (n = 8 Python and n = 8 wild type, Student's t test P = 0.12). (H) ATP level (mean ± SEM) after normalization to total protein level in extracts of tissue from Python and control mice at age 10 weeks. Student's t test P values are: heart +/+ (n = 7) vs. Py/+ (n = 6): P = 0.006; liver +/+ (n = 6) vs. Py/+ (n = 6): P = 0.80; brain +/+ (n = 6) vs. Py/+ (n = 5): P = 0.29. (I) Total adenine nucleotide pool (TAN) in extracts of tissue from Python and control mice at age 10 weeks. Values shown are mean ± SEM nmol ATP + ADP + AMP normalized to mg protein. Student's t test P values are: heart +/+ (n = 7) vs. Py/+ (n = 6): P = 0.008; liver +/+ (n = 6) vs. Py/+ (n = 6): P = 0.97; brain +/+ (n = 6) vs. Py/+ (n = 5): P = 0.31.